Title: The role of LARP6-mediated collagen mRNA transport in fibrosis
Project Summary: A common feature in fibrosis is the overproduction and deposition of extracellular matrix biomolecules outside the cell that form scar tissues with compromised structural qualities. The most abundant biomolecule in fibrotic tissues is typically collagen. While past drug treatment strategies have focused on the inflammatory pathways that lead to fibrosis, treating general inflammation leads to off target side effects. We propose that pursuing the molecular machinery that produces collagen will result in a more specific drug treatment with fewer side effects and with the ability to be integrated into a broad range of therapies treating extracellular matrix diseases. The long term goal of this proposal is to prevent fibrotic disease progression by disrupting the molecular machinery that produce collagen. The RNA binding protein LARP6 is known to transport collagen-encoding COL1A1 mRNA from the nucleus to the rough ER and it has been hypothesized that LARP6 localizes in clusters on the endoplasmic reticulum to induce cotranscriptional folding of the collagen triple helix. The overall objective of this proposal is to understand the molecular mechanisms that drive recognition of collagen mRNAs by LARP6. Our central hypothesis is that the LaM and RRM1 domains of LARP6 utilize conformational selection in the recognition and discrimination of collagen mRNAs. We further hypothesize that dynamic sampling of the tandem arrangement of the La and RRM domains allows LARP6 to accommodate a diverse set of mRNA ligands. Our rationale is that an understanding of LARP6-mediated mRNA transport will lead to the identification of novel therapeutic targets that can mitigate fibroproliferative diseases. To test our central hypothesis and achieve our overall objective, we propose the following specific aims: 1) Identify and characterize the binding kinetics and thermodynamics of mRNA targets of LARP6, 2) Determine the structural and dynamical contribution to LARP6/mRNA binding, 3) Characterize LARP6 mediated transport of Collagen mRNA in living fibroblasts. By utilizing novel in vivo RNA labeling strategies and live cell microscopy, we will be able to characterize RNP assembly, nuclear export and trafficking to the ER in living fibroblast cells. In the first aim, RNA targets of LARP6 will be identified using CLIP-seq and binding will be assessed with gel assays, analytical ultracentrifugation, and isothermal titration calorimetry. In the second aim, the structure and dynamics of the LARP6 LaM and RRM1 tandem domains alone and in the LARP6/mRNA complex. The third aim uses a live cell imaging approach to follow the formation and termination of LARP6/mRNA complexes in hepatic cells. The approach research is innovative in proposing LARP6 as a target for antifibrosis by apply progressive methods (CLIP-seq, integrated structural biology, and live cell microscopy) to characterize LARP6/mRNA interactions as the basis for future drug discovery studies. The results of this study will be significant as they are expected to have wide-reaching impact on treatment of a broad range of extracellular matrix diseases. It is essential that we understand the mode of RNA binding so that we can discern how LARP6 regulates specific genes and use this information to design antifibrotic drugs.